Dengue virus infections are serious health problems in many areas of the world. Dengue virus can cause two forms of disease, dengue fever (DF) and dengue hemorrhagic fever (DHF). While DF is a self-limiting febrile disease, DHF can lead to life-threatening complications. Laboratory diagnosis of dengue virus infection has depended mainly upon the isolation of dengue virus using mosquito cell cultures or inoculation of mosquitos, detection of anti-dengue antibody by IgM or IgG ELISA, and/or hemagglutination inhibition (HI) assays. Isolation of dengue virus is tedious and time consuming, however, and serological testing generally requires paired serum samples obtained several days or weeks apart, which are not always available.
Polymerase chain reaction (PCR) has the potential for sensitive, specific, and rapid detection of minute quantities of certain genetic material in clinical specimens, thus providing an attractive approach for the rapid diagnosis of dengue virus infection. Several methods of reverse transcriptase (RT)-PCR using different pairs of primers for dengue viruses and different approaches for the detection of amplification products have been previously reported. However, most of the published methods require more than 24 hours for analysis. Rapid RT-PCR methods for detection of dengue viremia have been reported, but they have not been shown to detect all four dengue serotypes in clinical specimens. The objective of several of these reports was to determine the serotype of dengue virus strain with which an individual was infected. RT-PCR followed by hybridization to serotype-specific probes, or RT-PCR followed by nested PCR, have been used for this purpose. Lanciotti et al., J. Clin. Microbiol., 30:545-551 (1992). Although these methods are sensitive, they still require considerable time and labor for use in patient management. Morita et al. describes a rapid and simple method using NP-40 and serotype-specific primers, in which RNA isolation, virus detection and typing can be done in a single reaction tube. Morita et al., J. Clin. Microbiol., 29:2107-2110 (1991); J. Med. Virol., 44:54-58 (1994).